Optical reader system and method for monitoring and correcting lateral and angular misalignments of label independent biosensors

ABSTRACT

An optical reader system and method are described herein that can detect a lateral and/or angular misalignment of one or more biosensors so that the biosensors can be properly re-located after being removed from and then reinserted into the optical reader system. In one embodiment, the biosensors are incorporated within the wells of a microplate.

CLAIMING BENEFIT OF CO-PENDING APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/789,900, filed Apr. 26, 2007, now pending, which is adivisional application of U.S. patent application Ser. No. 11/210,920,filed Aug. 23, 2005, now U.S. Pat. No. 7,629,173, which is acontinuation-in-part application of U.S. patent application Ser. No.11/027,547 filed Dec. 29, 2004, now U.S. Pat. No. 7,604,984. Thecontents of these documents are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an optical reader system and method fordetecting a lateral and/or angular misalignment of one or morebiosensors so that the biosensors can be properly re-located after beingremoved from and then reinserted into the optical reader system. In oneembodiment, the biosensors are incorporated within the wells of amicroplate.

2. Description of Related Art

A major challenge today is to design an optical reader system that canproperly re-locate a label independent detection (LID) microplate afterit is removed and then reinserted back into the optical reader system.In particular, what is needed is an optical reader system that candetect and correct a lateral and/or angular misalignment of are-positioned LID microplate. This need and other needs are addressed bythe optical reader system and method of the present invention.

BRIEF DESCRIPTION OF THE INVENTION

The present invention includes an optical reader system and method thatuses one or more fiducial markings (e.g., position sensors) on a LIDmicroplate to monitor and correct if needed any lateral and/or angularmisalignment of the microplate. In one embodiment, the method includesthe steps of: (a) placing the microplate onto a translation stage; (b)using one or more fiducial marking(s) on the microplate to determine afirst position of the microplate; (c) removing the microplate from thetranslation stage; (d) re-inserting the microplate back onto thetranslation stage; (e) using the fiducial marking(s) on the microplateto determine a second position of the microplate; (f) comparing thefirst position and the second position of the microplate; and (g) ifthere is a difference between the two positions, then addressing thelateral and/or angular misalignment of the microplate by: (1) moving thetranslation stage so that the microplate is located at or substantiallynear to the first position; or (2) not moving the microplate but insteadadjusting via software a measured reading (e.g., resonance wavelength)based upon the known position error and a known translation sensitivity.Likewise, steps (a)-(g) could be accomplished by using a stationaryholder for the microplate and instead the optical beams can be movedthat interrogate the stationary microplate. In another embodiment, theoptical reader system can be used to monitor and correct a lateraland/or angular misalignment of a biosensor (which has a fiducialmarking) that is not incorporated within a microplate.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the present invention may be had byreference to the following detailed description when taken inconjunction with the accompanying drawings wherein:

FIG. 1 is a block diagram of an optical reader system that is used tomonitor and correct a lateral and/or angular misalignment of amicroplate (or biosensor) in accordance with the present invention;

FIG. 2 is a graph that is used to help describe why the optical readersystem should monitor and correct the lateral and/or angularmisalignment of the microplate (or biosensor) in accordance with thepresent invention;

FIGS. 3-5, 6A and 6B are several graphs and diagrams used to helpdescribe one type of fiducial marking that can be formed on thebiosensor which enables the optical reader system to monitor and correctthe lateral and/or angular misalignment of the microplate (or biosensor)in accordance with the present invention;

FIGS. 6C and 6D are two diagrams used to help describe a second type offiducial marking that can be formed on the biosensor which enables theoptical reader system to monitor and correct the lateral and/or angularmisalignment of the microplate (or biosensor) in accordance with thepresent invention;

FIGS. 7A and 7B are two diagrams used to help describe a third type offiducial marking that can be formed on the microplate (or biosensor)which enables the optical reader system to monitor and correct thelateral and/or angular misalignment of the microplate (or biosensor) inaccordance with the present invention;

FIGS. 8-10 are three graphs which are used to help explain other usesfor the third type of fiducial marking in addition to enabling theoptical reader system to monitor and correct the lateral and/or angularmisalignment of the microplate (biosensor) in accordance with thepresent invention; and

FIG. 11 is a flowchart illustrating the steps of a method for monitoringand correcting a lateral and/or angular misalignment of a microplate (orbiosensor) in accordance with the present invention.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring to FIGS. 1-11, there are disclosed several diagrams and graphswhich are used to help describe the optical reader system 100 and method1100 of the present invention. As discussed below, the optical readersystem 100 is capable of performing two functions: (1) detecting abiological substance 124 (or a biomolecular binding event) on abiosensor 102; and (2) detecting and correcting any lateral and/orangular misalignment of the biosensor 102 which is caused by the removaland subsequent reinsertion of the biosensor 102 into the optical readersystem 100. Prior to discussing the second function, a brief descriptionis provided about how the optical reader system 100 can detect abiological substance 124 on the biosensor 102.

As shown in FIG. 1, the optical reader system 100 is used to interrogatea biosensor 102 (e.g., resonant waveguide grating (RWG) biosensor 102, asurface plasmon resonance (SPR) biosensor 102) to determine if abiological substance 124 is present on the biosensor 102. The opticalreader system 100 includes a light source 106 (e.g., lamp, laser, diode)that outputs an optical beam 104 which is scanned across the biosensor102. Typically, the biosensor 102 is moved so the optical beam 104 canbe scanned across the biosensor 102. Alternatively, the optical beam 104itself may be scanned with a mirror, galvanometer, electro-optic oracousto-optic scanner or other suitable adjustable optical element,across a stationary biosensor 102. While the optical beam 104 is scannedacross the biosensor 102, a detector 108 (e.g., spectrometer, CCD cameraor other optical detector) collects an optical beam 112 which isreflected from the biosensor 102. A processor 110 (e.g., DSP 110,computer 110) then processes the collected optical beam 112 to obtainand record raw spectral data 114 which is a function of a position (andpossibly time) on the biosensor 102. Thereafter, the processor 110analyzes the raw spectral data 114 to create a spatial map of resonantwavelength (peak position) data which indicates if a biologicalsubstance 124 is present on the biosensor 102.

In particular, the biosensor 102 makes use of changes in the refractiveindex at the sensor surface 126 that affect the waveguide couplingproperties of the emitted optical beam 104 and the detected optical beam112 to enable label-free detection of the biological substance 124(e.g., cell, molecule, protein, drug, chemical compound, nucleic acid,peptide, carbohydrate) on the superstrate 103 (sensing region) of thebiosensor 102. The biological substance 124 may be located within a bulkfluid that is deposited on the superstrate 103 (sensing region) of thebiosensor 102 and it is the presence of this biological substance 124that alters the index of refraction at the surface 126 of the biosensor102. Thus, to detect the biological substance 124, the biosensor 102needs to be at least probed with an optical beam 104 and then areflected optical beam 112 received at the detector 108 is analyzed todetermine if there are any changes (˜1 part per million) in therefractive index caused by the presence of the biological substance 124.In one embodiment, the top surface 126 may be coated with biochemicalcompounds (not shown) that only allow surface attachment of specificcomplementary biological substances 124 which enables a biosensor 102 tobe created that is both highly sensitive and highly specific. In thisway, the optical reader system 100 and biosensor 102 may be used todetect a wide variety of biological substances 124. And, if multiplebiosensors 102 are arranged in array like in a microplate 126 then theymay be used to enable high throughput drug or chemical screeningstudies. For a more detailed discussion about the detection of abiological substance 124 (or a biomolecular binding event) using thescanning optical reader system 100, reference is made to theaforementioned U.S. patent application Ser. No. 11/027,547.

It is well known that when an optical beam 104 is used to interrogate abiosensor 102, then the resonance wavelength often has an undesirabledependence upon the exact spatial location at which the optical beam 104strikes the biosensor 102. The undesirable variation of the resonancewavelength is often caused by the non-homogeneity of the biosensor 102which can be attributable to variations in the thickness of thewaveguide and/or to variations in the grating period (for example). Infact, a typical variation in the resonance wavelength can be as high as3 pm per micron. Thus, if one desires to remove and replace thebiosensor 102 from the optical reader 100 during the course of anexperiment, the biosensor 102 needs to be repositioned to a highaccuracy to prevent wavelength shifts induced by translation fromoverwhelming those wavelength shifts from biochemical binding. Theimpact, in terms of wavelength shift Δλ of such a translationsensitivity upon the measurement is thus

${\Delta\lambda} = {{\frac{\lambda}{x} \cdot \Delta}\; {x.}}$

Here Δλ/dx is the translation sensitivity (pm/μm) and Δx is thedisplacement (μm) of the biosensor 102 between measurements. Thisformula makes apparent two ways of reducing the impact oftranslation: 1) reduce the translation sensitivity, Δλ/dx, by carefuldesign of the biosensor 102 and/or the optical reader system 100; or 2)reduce the amount of displacement Δx that occurs between measurements.

To reduce the translation sensitivity, the scanning optical readersystem 100 can be used to average these spatial fluctuations in theresonance wavelength. This has been shown to decrease the translationalsensitivity by an order of magnitude to around 0.3 pm per micron. FIG. 2is a graph that shows the typical shape of the resonance wavelength(spectral shift) that can be obtained when scanning one 3 mm longbiosensor 102 in one direction with a 100 μm diameter optical beam. Itshould be appreciated that the use of a “larger” optical beam 104 canhelp even more by further averaging down high spatial frequencyvariations. Although, a resonance wavelength translation sensitivity of0.3 pm per micron works well in many applications, such a sensitivitycan still be of great concern for systems attempting to detect smallbiomolecular binding events. Such small binding events can requireresonant wavelength measurement accuracies of better then 0.05 pm. Toaddress this problem one can minimize the translation induced wavelengtherror by ensuring that the biosensors 102 are properly positioned withinthe optical reader system 100. This is done by the second function ofthe optical reader system 100.

A detailed discussion is provided next about three different ways theoptical reader system 100 can make sure that the biosensors 102 areproperly positioned therein. Basically, the optical reader system 100can detect and correct a lateral and/or an angular misalignment of thebiosensor(s) 102 (microplate 126) by using anyone or a combination ofthree different types of fiducial markings which can be located oneither the biosensor 102 or the microplate 126. The first type offiducial marking is the edge of the measurement diffraction grating onthe biosensor 102 (see FIGS. 3-5 and 6A-6B). The second type of fiducialmarking is a non-responding line 602, 602 a and 602 b located on themeasurement diffraction grating of the biosensor 102 (see FIGS. 6C-6D).And, the third type of fiducial marking is a fiducial diffractiongrating 702 (position sensor 702) that is separate from the measurementdiffraction grating on the biosensor 102 (see FIGS. 7A and 7B). In yetanother embodiment, the fiducial marking can be a coating (localmetallic, dielectric coating) that is applied to a biosensor 102 ormicroplate 126. This coating would have a sufficient reflectivitycontrast so it could be detected by the optical reader system 100.

In the first way, the optical reader system 100 scans a biosensor 102and uses the resulting raw spectral data 114 to create a spatial map ofreflected power that enables one to precisely locate the edge of thegratings in the biosensor 102. FIG. 3 is a graph that shows the typicalshape to the power evolution of the resonance wavelength when theoptical reader system 100 scans a square biosensor 102. To determine theedges of the biosensor 102, various edge detection algorithms can beused. As an example, FIG. 4 is a graph that shows the result of applyinga derivative filter on the power profile shown in FIG. 3. By detectingthe centroids of the positive and negative peaks of the differentiatedsignal, one can accurately determine the position of the biosensor 102.Again, in this case the fiducial marking is the edge of the measurementdiffraction grating on the biosensor 102.

To estimate the repeatability of this type of position measurement, asquare biosensor 102 was scanned 500 hundred times. The position of thedetected edge then was measured with respect to the position encoderdata on a translation stage 128 which supported the biosensor 102 (ormicroplate 126) (see FIG. 1). Results of this test are shown in FIG. 5.The typical standard deviation of the measurements is in the range of0.25 microns, which is in fact very close to the resolution of theencoder on the translation stage 128 (see FIG. 1).

This and other types of position measurements are important, becausewhen a microplate 126 which contains an array of biosensors 102 isremoved from and then reinserted into the optical reader system 100, oneessentially loses track of the absolute translational position of themicroplate 126. However, upon reinsertion, when the optical beam 104 isscanned across the microplate 126, by detecting the location where theedges of the grating(s) occur on the biosensor(s) 102, one can“recalibrate” the translation stage 128 (e.g., linear stages 128) so itcan very precisely move the microplate 126 back to the same position themicroplate 126 was in before it was removed from the optical readersystem 100. For a more detailed discussion about how the optical readersystem 100 can detect the edges of a measurement diffraction grating ina biosensor 102, reference is made to the aforementioned U.S. patentapplication Ser. No. 11/027,547.

Additionally, one may use various edge detection concepts to monitor thetwo dimensional (2D) lateral position of the microplate 126 (see FIGS.6A-6D). In one such edge detection concept, a square biosensor 102 isscanned in both an x direction and a y direction to determine thelateral 2D position of the microplate 126 (see FIG. 6A). In another edgedetection technique, one can scan a triangular biosensor 102 where thex-position is given by the position of the first edge detection and they-position is given by the distance measured between the two edgesdetections (see FIG. 6B).

In yet another other edge detection concept, one can use the second typeof fiducial marking(s) 602 which are non-responding line(s) 602 locatedon the biosensor 102 to monitor the lateral 2D position of themicroplate 126 (see FIGS. 6C-6D). In one example, the biosensor 102 hasa design as shown in FIG. 6C where a non-responding line 602 was madediagonally across the biosensor 102. This diagonal non-responding line602 enables one to estimate both the x and y positions of the biosensor102 with a single 1-dimensional beam scan. In particular, when usingsuch a diagonal non-responding line 602 the rising edge of a power vs.position trace is used to determine the x-position and the differencebetween the rising and falling edges is used to determine they-position. In yet another example shown in FIG. 6D, the biosensor 102has two off-center non-responding lines 602 a and 602 b that are set atthe edge of the biosensor 102 which allows one to also use the centerportion of the grating to detect a biological substance 124 (or abiomolecular binding event). An advantage of the last example is thatone can put fiducial markings 602 a and 602 b on all of the biosensors102 which allows one to obtain more data that can be averaged to improvethe re-positioning accuracy. However, the drawback of this example isthat a complete measurement requires two scanning steps, one scanningstep for the position measurement and one scanning step for thebiochemical measurement itself. It should be noted that non-respondinglines 602, 602 a and 602 b can be generated by having some areas withouta diffraction grating or without a waveguide.

Referring now to the third type of fiducial marking, the optical readersystem 100 in this case scans a fiducial diffraction grating 702(position sensor 702) which is preferably located on a microplate 126(see FIGS. 7A and 7B). As shown in FIGS. 7A and 7B, the optical readersystem 100 can interrogate the fiducial diffraction gratings 702 whichare relatively close to the biosensor 102. Then, in real time measurethe position of the microplate 126 a and 126 b and if needed make thetranslation corrections before the interrogation beam 104 reaches thebiosensor 102. This allows continuous scanning with real time positioncorrection.

As can be seen in the exemplary microplates 126 a and 126 b shown inFIGS. 7A and 7B, one can put a fiducial diffraction grating 702 and abiosensor 102 in each measurement well 704 a (see FIG. 7A). Or, one canput a fiducial diffraction grating 702 at the beginning and at the endof the microplate plate 126 b (see FIG. 7B). In the last case, thefiducial diffraction gratings 702 are located outside the measurementwells 704 b and will be in contact with air or with the glue that holdstogether the microplate 126. The design of these particular fiducialdiffraction gratings 702 in terms of a grating period should beoptimized to generate a resonance wavelength close to the one that wouldbe generated if the fiducial diffraction gratings 702 were in contactwith the aqueous buffer solutions likely to be used in the wells. Thisis because the global spectral range of the optical reader system 100 islimited by the spectral width of the light source 106 and detector 108,and it is important to keep the resonance within the operational band ofthis source/detector system 100.

An advantage of having multiple fiducial diffraction gratings 702 acrossthe microplate 126 a and 126 b is that one can average the data andobtain a better measurement accuracy. Another advantage of havingmultiple fiducial diffraction gratings 702 on a microplate 126 a and 126b is that it allows one to monitor thermal dilatations of the microplate126 a and 126 b. To measure thermal dilations of the microplate 126 aand 126 b one can optically scan the microplate 126 a and 126 b andrecord the locations of the fiducial diffraction gratings 702 (F1, F2,F3 . . . ) (or other types of fiducial markings). Then, after some timeand possibly a temperature change, one may rescan the microplate 126 aand 126 b and again record the locations of the same fiducialdiffraction gratings 702 (F1, F2, F3 . . . ) (or other types of fiducialmarkings). If the microplate 126 a and 126 b has grown or shrunk due totemperature change, then the relative locations of the fiducialdiffraction gratings 702 (or other types of fiducial markings) will havechanged (i.e., Δ₂₁=F2−F1 will have changed, and Δ₃₁=F3−F1 will havechanged . . . ).

In an alternative embodiment, the fiducial diffraction gratings 702 andthe measurement diffraction gratings can have different resonancewavelengths. To have different resonance wavelengths, the fiducialdiffraction gratings 702 and the measurement diffraction gratings can bemade with different grating periods. Or, they can be made withwaveguides that have different thicknesses. In this embodiment, theresonance wavelengths can be detected by measuring the evolution of thepower of the two peaks corresponding to the different gratings areas.Then, the edge detection can be made based on the relative power of bothpeaks.

In yet another embodiment, the fiducial gratings 702 can includefeatures that are perpendicular to the scanning direction of the opticalbeam and other features that are at a certain angle such as 45 degreeswith respect to the scanning direction. In this way, one can determinemisalignments in both directions.

A discussion is provided next about several other uses of the fiducialdiffraction gratings 702 (position sensors 702) in addition to their usein helping with the repositioning of the biosensor 102 or microplate126. When the fiducial diffraction gratings 702 are not in contact withthe liquid that is measured in the wells of the microplate 126, thenthose fiducials are completely isolated and their resonance wavelengthis affected only by disturbing external effects such as temperaturevariations or angular misalignments. As a result, one can use thefiducial diffraction gratings 702 to monitor those external effects asfollows:

1. Angular Monitoring—FIG. 8 shows the typical wavelength shift that canbe measured as a function of the incidence angle when interrogatingbiosensors 102 at normal incidence with single mode fibers. As can beseen, the angular sensitivity is in the range of 10 pm/mRd which canmake the angle monitoring very critical. One way that this angularvariation can be monitored is to interrogate the fiducial diffractiongratings 702 by using a multimode fiber instead of a single mode fiberfor the light injection. Indeed, as shown on FIG. 9, when thisconfiguration was tested we obtained angular sensitivities in the rangeof 432 pm/mRd which is an order of magnitude greater than thesensitivity that obtained with the single mode fibers. So, by comparingthe resonance wavelength measured with a multimode fiber and the onemeasured with the single mode fiber, one can better deduce any angularmisalignment of the microplate 126 after reinserting it into the reader100 by using the multimode fiber.

2. Temperature Gradient Monitoring—FIG. 10 is a graph that shows thetemperature sensitivity of an interrogated fiducial diffraction grating702 when the temperature cools by 21° C. As can be calculated from thewavelength changes of the curves shown, the sensitivity coefficients are−10 pm/° C. for TE mode and −26 pm/° C. for the TM mode. Therefore,temperature changes of as small as 0.01° C. can perturb the measuredresonance wavelengths by 0.26 pm, which is of significance for smallbiomolecular binding events. Additionally, even if in-well referencingis used (see U.S. patent application Ser. No. 11/027,509 entitled“Method for Creating a Reference Region and a Sample Region on aBiosensor and the Resulting Biosensor and U.S. patent application Ser.No. 11/027,547 entitled “Spatially Scanned Optical Reader System andMethod for Using Same”) temperature gradients, and in particular changesin temperature gradients, inside wells may still be large enough toinduce resonant wavelength shifts of concern. Assuming that the fiducialdiffraction gratings 702 are in contact with glue, then the temperaturevariation is the major parameter that makes the resonance wavelengthfluctuate over time. With this knowledge one can then use the wavelengthfluctuations measured across the fiducial diffraction gratings 702 onthe microplate 126 a and 126 b to deduce the temperature gradientfluctuations and check that they are under acceptable levels.

From the foregoing, it can be readily appreciated by those skilled inthe art that the present invention also includes a method 1100 formonitoring and correcting if needed any lateral and/or angularmisalignment of the microplate 126. As shown in the flowchart of FIG.11, the method 1100 includes the steps of: (a) place microplate (withone or more fiducial markings) on holder which in one embodiment is thetranslation stage 128 and in another embodiment is a stationary holder(not shown) (step 1102); (b) using one or more fiducial markings on themicroplate 126 to determine a first position of the microplate 126 (step1104); (c) removing the microplate 126 from the holder (step 1106); (d)reinserting the microplate 126 back onto the holder (step 1108); (e)using the fiducial marking(s) on the microplate 126 to determine asecond position of the microplate 128 (step 1110); (f) comparing thefirst position and the second position of the microplate 126 (step1112); (g) if there is a difference between the two positions, thenaddressing the lateral and/or angular misalignment of the microplate 126(step 1114) by: (1) moving the translation stage 128 so that themicroplate 126 is located at or substantially near to the first position(step 1114 a); or moving the optical beams 104 so that the microplatewhich is on the stationary holder appears to be in the first position(step 1114 b); or (3) not moving the microplate 126 or the optical beams104 but instead adjusting via software a measured interrogation reading(e.g., resonance wavelength) based upon the known position error and aknown translation sensitivity (step 1114 c); and (h) if there is nodifference (or no substantial difference) between the two positions,then interrogate the microplate 126 while it is in the second position(step 1116).

It should be noted that the term angular misalignment as used above isthe skew that is caused by the microplate 126 being rotated in the Zaxis if the X&Y are the lateral axis. Alternatively, it should be notedthat an angular misalignment can also be caused if one performs a“skewed” scan across the microplate 126 where one simultaneously movesthe X&Y motion stages in a coordinated skewed motion.

It should also be noted that in most of the drawings herein, were madebased on the assumption that the sensor is spectrally interrogated. Thismeans that the sensor is interrogated at a fixed incidence angle with abroad spectral source and that the wavelength is detected in thereflected beam. The source is then a broad spectral source and thedetector is a wavelength sensitive detector such as a spectrometer.However, it should be appreciated that the principle of the presentinvention can also be extended to an angular interrogation approachwhere the biosensor is interrogated with monochromatic light and then aresonant angle is detected in the reflected beam.

Furthermore, it should be noted that there are configurations of thepresent invention that do not need to use scanning to position,re-position and/or interrogate the biosensor 102. One such non-scanningsystem involves the use of a vision system. The vision system wouldcreate an image of the biosensor(s) 102, the optical beams 104, and/orthe fiducials on a position sensitive detector (e.g., CCD camera). And,this vision system could make use of the fiducials by looking at theposition of the fiducials imaged on the CCD camera and then make theappropriate adjustments.

Although multiple embodiments of the present invention have beenillustrated in the accompanying Drawings and described in the foregoingDetailed Description, it should be understood that the invention is notlimited to the embodiments disclosed, but is capable of numerousrearrangements, modifications and substitutions without departing fromthe spirit of the invention as set forth and defined by the followingclaims.

1. A method for detecting and correcting a misalignment of a microplatein an optical reader system, said method comprising the steps of:placing said microplate onto a holder; using a fiducial marking on saidmicroplate to determine a first position of said microplate; removingsaid microplate from said holder; re-inserting said microplate back ontosaid holder; using said fiducial marking on said microplate to determinea second position of said microplate; comparing the first position andthe second position of said microplate; and if there is a differencebetween the two positions, then addressing the lateral and/or angularmisalignment of said microplate such that the re-inserted microplate ispositioned to be located at or substantially near to the first position,wherein said fiducial marking on said microplate is a coating.
 2. Themethod of claim 1, wherein said addressing step includes the step ofmoving said holder so that the microplate is located at or substantiallynear to the first position.
 3. The method of claim 1, wherein saidaddressing step includes the step of moving one or more optical beamsused to interrogate said fiducial marking so that the microplate islocated at or substantially near to the first position.
 4. The method ofclaim 1, wherein said addressing step includes the step of usingsoftware to adjust a measured interrogation reading based upon the knownposition error and a known translation sensitivity.
 5. The method ofclaim 1, wherein said step of using the fiducial marking on saidmicroplate to determine either the first position or the second positionof said microplate includes: generating an optical beam; scanning theoptical beam across said fiducial marking on said microplate; collectingthe scanned optical beam which is reflected from said fiducial markingon said microplate; processing the collected optical beam to determineeither the first position or the second position of said microplate as afunction of a position of said holder; and recording either the firstposition or the second position of said microplate as a function of theposition of said holder.
 6. The method of claim 1, further comprisingthe step of determining whether or not a biological substance is presentor a biomolecular event occurred on a surface of a measurementdiffraction grating within a well in said microplate.
 7. The method ofclaim 6, wherein said determination of the second position of saidmicroplate and said determination of whether or not a biologicalsubstance is present or a biomolecular event occurred within the well insaid microplate are performed in one optical beam scanning step.
 8. Themethod of claim 6, wherein said determination of the second position ofsaid microplate and said determination of whether or not a biologicalsubstance is present or a biomolecular event occurred within the well insaid microplate are performed in two optical beam scanning steps.
 9. Themethod of claim 1, further comprising the step of scanning multiplefiducial markings on said microplate to measure thermal dilations ofsaid microplate.
 10. The method of claim 1, further comprising the stepof scanning the fiducial marking which is a fiducial diffraction gratingto determine a temperature gradient.
 11. The method of claim 1, furthercomprising the step of scanning the fiducial marking which includesfeatures that are at one angle to a scanning direction of an opticalbeam and features that are at a second angle to the scanning directionof the optical beam which enables one to determine misalignments if anyin two directions.
 12. An optical reader system comprising: a holder forsupporting a biosensor; a light source for creating an optical beamwhich is scanned across a fiducial marking associated with thebiosensor; a detector for collecting the scanned optical beam which isreflected from the fiducial marking associated with the biosensor; aprocessor for analyzing the collected optical beam and determining aposition of the biosensor; and said processor for addressing a lateraland/or an angular misalignment of the biosensor, if needed.
 13. Theoptical reader system of claim 12, wherein said fiducial marking is afiducial diffraction grating which is located away from a measurementdiffraction grating that is associated with the biosensor.
 14. Theoptical reader system of claim 12, wherein said fiducial markingincludes features that are at one angle to a scanning direction of theoptical beam and features that are at a second angle to the scanningdirection of the optical beam which enables one to determinemisalignments if any in two directions.
 15. The optical reader system ofclaim 12, wherein said biosensor is incorporated within a microplate.16. A microplate comprising: a frame including a plurality of wellsformed therein, each well incorporating a biosensor that includes: asubstrate; a measurement diffraction grating; and a waveguide film; andsaid frame further includes at least one fiducial marking locatedthereon which is used to help determine a position of the biosensors.17. The microplate of claim 16, wherein one of said at least onefiducial marking is located outside the wells.
 18. The microplate ofclaim 16, wherein said fiducial marking is: a grating area having adifferent resonance wavelength; a fiducial diffraction grating; or acoating.